Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Neovascularization during tumorigenesis supplies oxygen and nutrients to proliferative tumor cells, and serves as a conduit for migration. Targeting oncogenes involved in angiogenesis is needed to treat organ-confined and locally advanced ESCC. Although the phospholipase C epsilon-1 (PLCE1) gene was originally identified as a susceptibility gene for ESCC, how PLCE1 is involved in ESCC is unclear. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used to measure the methylation status of the PLCE1 promoter region. To validate the underlying mechanism for PLCE1 in constitutive activation of the NF-κB signaling pathway, we performed studies using in vitro and in vivo assays and samples from 368 formalin-fixed esophageal cancer tissues and 215 normal tissues with IHC using tissue microarrays and the Cancer Genome Atlas dataset. We report that hypomethylation-associated up-regulation of PLCE1 expression was correlated with tumor angiogenesis and poor prognosis in ESCC cohorts. PLCE1 can activate NF-κB through phosphoinositide-phospholipase C-ε (PI-PLCε) signaling pathway. Furthermore, PLCE1 can bind p65 and IκBα proteins, promoting IκBα-S32 and p65-S536 phosphorylation. Consequently, phosphorylated IκBα promotes nuclear translocation of p50/p65 and p65, as a transcription factor, can bind vascular endothelial growth factor-C and bcl-2 promoters, enhancing angiogenesis and inhibiting apoptosis in vitro. Moreover, xenograft tumors in nude mice proved that PLCE1 can induce angiogenesis, inhibit apoptosis, and increase tumor aggressiveness via the NF-κB signaling pathway in vivo. Our findings not only provide evidence that hypomethylation-induced PLCE1 confers angiogenesis and proliferation in ESCC by activating PI-PLCε-NF-κB signaling pathway and VEGF-C/Bcl-2 expression, but also suggest that modulation of PLCE1 by epigenetic modification or a selective inhibitor may be a promising therapeutic approach for the treatment of ESCC.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressively malignant tumors with dismal prognosis. Profilin 2 (PFN2) is an actin-binding protein that regulates the dynamics of actin polymerization and plays a key role in cell motility. Recently, PFN2 have emerged as significant regulators of cancer processes. However, the clinical significance and biological function of PFN2 in ESCC remain unclear. PFN2 protein expression was validated by immunohistochemistry (IHC) on tissue microarray from Chinese Han and Kazakh populations with ESCC. The associations among PFN2 expression, clinicopathological features, and prognosis of ESCC were analyzed. The effects on cell proliferation, invasion and migration were examined using MTT and Transwell assays. Markers of epithelial–mesenchymal transition (EMT) were detected by Western blot analysis. Compared with normal esophageal epithelium (NEE), PFN2 protein expression was markedly increased in low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and ESCC, increased gradually from LGIN to ESCC, and finally reached high grade in HGIN in the Han population. Similarly, PFN2 protein was more overexpressed in ESCC than in NEE in the Kazakh population. The results of Western blot analysis also showed that PFN2 expression was significantly higher in the ESCC tissue than in a matched adjacent non-cancerous tissue. PFN2 expression was positively correlated with invasion depth and lymph node metastasis. High PFN2 expression was significantly correlated with short overall survival (OS) (P = 0.023). Cox regression analysis revealed that PFN2 expression was an independent prognostic factor for poor OS in ESCC. Downregulation of PFN2 inhibited, rather than proliferated, cell invasion and migration, as well as induced an EMT phenotype, including increased expression of epithelial marker E-cadherin, decreased mesenchymal marker Vimentin, Snail, Slug and ZEB1, and morphological changes in ESCC cells in vitro. Our findings demonstrate that PFN2 has a novel role in promoting ESCC progression and metastasis and portending a poor prognosis, indicating that PFN2 could act as an early biomarker of high-risk population. Targeting PFN2 may offer a promising therapeutic strategy for ESCC treatment.

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with dismal prognosis and high incidence and mortality in Kazakh population. MiR-34a, a direct p53 target gene, possesses tumor-suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. The reduced expression of miR-34a by methylation in various cancers has been reported. To determine whether aberrant miR-34a methylation occurs in esophageal cancer, the DNA methylation of 23 CpGs sites in the miR-34a promoter was quantitatively analyzed in relation to the translation initiation site by MALDI -TOF mass spectrometry in 59 ESCC tissues and 34 normal tissues from the Kazakh population. Real-time PCR was used to detect the inhibition of miR-34a expression levels and to evaluate their association with methylation. We found that miR-34a is more frequently methylated in ESCC (0.133 ± 0.040) than in controls (0.066 ± 0.045, P < 0.01). A nearly two-fold increase in miR-34a expression for the hypomethylated promoter was found in normal esophageal tissues than ESCC with hypermethylation (P <0.0001), pointing to a negative relationship between miR-34a CpG sites methylation and expression(r = −0.594, P = 0.042). The hypermethylation of miR-34a CpG_8.9 was associated with the advanced UICC stage III/IV of the esophageal cancers, and the hypermethylation of CpG_8.9 and CpG_5 of miR-34a was significantly correlated with lymph node metastasis. Our findings suggest that miR-34a is involved in the etiology of ESCC and that hypermethylated miR-34a is a potential biomarker for ESCC diagnosis and prognosis. Moreover, targeting miR-34a methylation by demethylating agents may offer a novel strategy for anticancer therapy of ESCC.

Esophageal squamous cell carcinoma (ESCC) is a highly lethal cancer, and its underlying molecular mechanisms are poorly understood. Recent large-scale genome-wide association studies in Chinese Han populations have identified an ESCC susceptibility locus within the SLC39A6 gene. Here, we sought to explore the expression and biological function of SLC39A6 in ESCC. Multiethnic validation of SLC39A6 protein expression was performed in different cohorts of patients from Chinese Han and Kazakh populations in the Xinjiang region by immunohistochemistry. The associations among SLC39A6 expression, clinicopathological parameters, and prognosis outcomes of ESCC were analyzed. And the effects of SLC39A6 silencing by siRNA on cell proliferation, apoptosis, and invasiveness, as well as the proteins involved in epithelial-to-mesenchymal transition (EMT) of esophageal cancer cells, were studied. SLC39A6 protein expression increased progressively from normal esophageal epithelium (NEE) to low-grade intraepithelial neoplasia to ESCC, and finally reached the highest in high-grade intraepithelial neoplasia from Han ethnic. Similarly, SLC39A6 protein was significantly overexpressed in Kazakh ethnic ESCC compared with that in NEE. Increased expression of SLC39A6 was found to be closely correlated with histological grade and early Tumor-Node-Metastasis stage I/II. High tumorous SLC39A6 expression was significantly correlated with shorter overall survival (OS). Cox regression analysis confirmed that SLC39A6 expression was an independent prognostic factor for poor OS in ESCC. Experimentally, the suppression of SLC39A6 expression promoted ESCC cell apoptosis but abrogated proliferation and invasion, and induced an EMT phenotype that included enhanced expression of E-cadherin, loss of vimentin, and morphological changes in ESCC cells in vitro. Combined, our findings highlight a tumor-promoting role for SLC39A6 in ESCC, suggesting that SLC39A6 could serve as an early detector of high-risk subjects and prognostic biomarker. The targeting of SLC39A6 might be a potential therapeutic strategy for blocking ESCC.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest malignant diseases. Multiple studies with large clinic-based cohorts have revealed that variations of phospholipase C epsilon 1 (PLCE1) correlate with esophageal cancer susceptibility. However, the causative role of PLCE1 in ESCC has remained elusive. Here, we observed that hypomethylation-mediated upregulation of PLCE1 expression was implicated in esophageal carcinogenesis and poor prognosis in ESCC cohorts. PLCE1 inhibited cell autophagy and suppressed the protein expression of p53 and various p53-targeted genes in ESCC. Moreover, PLCE1 decreased the half-life of p53 and promoted p53 ubiquitination, whereas it increased the half-life of mouse double minute 2 homolog (MDM2) and inhibited its ubiquitination, leading to MDM2 stabilization. Mechanistically, the function of PLCE1 correlated with its direct binding to both p53 and MDM2, which promoted MDM2-dependent ubiquitination of p53 and subsequent degradation in vitro. Consequently, knockdown of PLCE1 combined with transfection of a recombinant adenoviral vector encoding wild-type p53 resulted in significantly increased levels of autophagy and apoptosis of esophageal cancer in vivo. Clinically, the upregulation of PLCE1 and mutant p53 protein predicted poor overall survival of patients with ESCC, and PLCE1 was positively correlated with p53 in ESCC cohorts. Collectively, this work identified an essential role for PLCE1- and MDM2-mediated ubiquitination and degradation of p53 in inhibiting ESCC autophagy and indicates that targeting the PLCE1–MDM2–p53 axis may provide a novel therapeutic approach for ESCC. Significance: These findings identify hypomethylation-mediated activation of PLCE1 as a potential oncogene that blocks cellular autophagy of esophageal carcinoma by facilitating the MDM2-dependent ubiquitination of p53 and subsequent degradation.